The flavin-containing monooxygenase gene family contains five known members. The drug metabolizing enzymes have wide, overlapping substrate specificities and participate in the oxidative metabolism of numerous drugs, pesticides and other environmental chemicals. Of these enzymes, the one with the most unique properties is FMO5. This isoform was cloned from rabbit and found not to metabolize the substrates usually associated with the FMO -- methimazole, thiobenzamide and dimethyaniline. Substrates identified for FMO5 were confined to a few short-chain primary amines like n-octylamine. This limited substrate specificity is not characteristic of other FMOs. It was suggested that the restricted activity of FMO5 was a function of the enzyme from rabbit and might not apply to FMO5 orthologs. However, we have now cloned FMO5 for the human and guinea pig and found that the properties of these two orthologs are the same as those of the enzyme from rabbit. Of the isoforms of FMO identified, FMO4 is the least understood. This isoform differs from the others in that its derived sequence contains a C-terminal extension of 24 amino acids. Attempts to express FMO4 in E. coli, yeast and COS-1 cells have, until recently, proven unsuccessful. This has been the case for the cDNA from either rabbits or humans. We have overcome this problem altering the position of the stop codon of the human cDNA and eliminating the C-terminal extension. The modified cDNA is highly expressed in E. coli. Modification of FMO3 to produce an elongated coding region similar to that of FMO4 markedly reduces the ability of FMO3 to be expressed in E. coli. We are attempting to learn whether or not these finding have any relevance to mammalian systems. Also, we are characterizing expressed FMO4 and purifying sufficient enzyme for antibody production. Expression of FMO4 in mammalian tissue has not been verified. However, results of PCR experiments indicate that the only FMO transcript in brain RNA is that of FMO4.